Synergistic interaction between sphingomyelin and gemcitabine potentiates ceramide-mediated apoptosis in pancreatic cancer

We have examined the mechanism by which sphingomyelin (SM) enhances chemotherapy in human pancreatic cancer cells, focusing on the correlation between ceramide metabolism and apoptosis. Dose response curves for gemcitabine in the absence or presence of 0.2 mg/mL SM provided IC50 values of 78.3 +/- 13.7 and 13.0 +/- 3.0 nmol/L, respectively. The cytotoxic effect of the combined treatment was synergistic (combination index = 0.36). Using annexin-V staining, the percentage of apoptotic cells was 3.6 +/- 2.6% for the untreated cells, 6.5 +/- 3.8% for the 0.2 mg/mL SM-treated cells, and 19.9 +/- 12.9% for the 100 nmol/L gemcitabine-treated cells, but increased significantly to 42.1 +/- 12.7% with the combined treatment (P < 0.001, compared with gemcitabine-treated group). The percentage of cells losing mitochondrial membrane potential followed a similar trend. The ceramide content of untreated and gemcitabine-treated cells was not significantly different (0.46 +/- 0.29 and 0.59 +/- 0.34 pmol ceramide/nmole PO4). However, when 0.2 mg/mL SM was added, ceramide levels were 1.09 +/- 0.42 and 1.58 +/- 0.55 pmol ceramide/nmol PO4, for the SM alone and SM with gemcitabine-treated cells, respectively (P = 0.038). Acidic SMase was activated by exposure to gemcitabine but not SM, whereas the activities of neutral SMase and glycosylceramide synthase did not change with either gemcitabine or SM. The data are consistent with gemcitabine-induced activation of acidic SMase and indicate that the addition of SM can yield increased production of ceramide, mitochondrial depolarization, apoptosis, and cell death. Because SM by itself is relatively nontoxic, addition of this lipid to agents that induce apoptosis may prove useful to enhance apoptosis and increase cytotoxicity in cancer cells.